Nucleic acid molecules which encode proteins having fructosyl transferase activity and methods for producing long-chain inulin

ABSTRACT

Nucleic acid molecules which encode proteins having fructosyl transferase activity and methods for producing long-chain inulin Nucleic acid molecules are described encoding proteins with the enzymatic activity of a fructosyl transferase. These enzymes are fructosyl transferases (FFT). Moreover, vectors and host cells are described containing the nucleic acid molecules of the invention, in particular transformed plant cells, plant tissue and plants regenerable therefrom, which express the described FFT. Furthermore, methods for the production of long-chain inulin by using the described proteins, hosts, in particular the plant cells and/or FFT produced by them, are described.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of international applicationPCT/EP98/07115, filed Nov. 6, 1998, which designated the United States.

BACKGROUND OF THE INVENTION

The present invention relates to nucleic acid molecules encodingproteins with the enzymatic activity of a fructosyl transferase (FFT).The invention also relates to vectors containing such nucleic acidmolecules as well as to host cells transformed with said nucleic acidmolecules, in particular plant cells, plant tissue and plants. Moreover,methods for the production of transgenic plants are described whichsynthesize long-chain inulin due to the introduction of nucleic acidmolecules encoding an FFT. The present invention also relates to methodsof producing FFT and to the production of long-chain inulin in varioushost organisms, in particular plants, as well as to in vitro methods forproducing long-chain inulin by means of the FFT of the invention. Thepresent invention further relates to the host cells of the invention andto the inulin obtainable by the processes of the present invention.

Water-soluble, linear polymers allow for a variety of applications, forexample for increasing the viscosity in aqueous systems, as detergents,as suspending agents or for speeding up sedimentation, for complexingand, however, also for binding water. Polymers which are based onsaccharides, such as fructosyl polysaccharides, are particularlyinteresting raw materials as they are biodegradable. Apart from theirapplication as regenerable raw materials for the industrial productionand processing, fructosyl polymers are also to be considered asadditives in foodstuffs, for example as sweeteners. For various uses,polymers with varying chain-lengths are needed. Whereas short- andmedium-chain polymers are particularly preferred in the food processingindustry, polymers with a high degree of polymerization (DP) are neededfor technical uses, such as the production of surfactants.

So far only methods for producing long-chain fructan polysaccharides inplants have been described in which fructosyl transferases of bacterialorigin are expressed. Most bacterial fructosyl transferases synthesizelevan, a β-2,6 linked fructosyl polymer which has numerousβ-2,1-branchings. Due to its numerous branchings levan has decisivedisadvantages when it comes to technical processing and is thereforeconsiderably less significant as a technical raw material then inulin.Up to now, only one bacterial gene is known, the gene product of whichis involved in the synthesis of inulin, namely the ftf gene fromStreptococcus mutans. It is in principle possible to express the gene inplants if the gene has previously been genetically engineered. However,the inulin yield obtained from transgenic plants is so low that theeconomic utilization of the transgenic plants is out of question.

Furthermore, a method for producing transgenic plants expressingfructosyl transferases from Helianthus tuberosus is known. Theexpression of these genes in transgenic plants leads to the productionof inulin with an average degree of polymerization of DP=6 to DP=10.Polymers with this degree of polymerization may not be referred to aslong-chain inulin. Inulin with an average DP=6 to DP=10 is unsuitablefor most technical uses.

Methods for an economic production of long-chain inulin in plants or forsynthesizing enzymes for the production of long-chain inulin are notknown.

PCT/US89/02729 describes the possibility of synthesizing carbohydratepolymers, in particular dextran or polyfructose, in transgenic plantcells, specifically in the fruits of transgenic plants. In order toproduce plants modified in such a way, the use of levan sucrases frommicroorganisms, in particular from Aerobacter levanicum, Streptococcussalivarius and Bacillus subtilis, or of dextran sucrases fromLeuconostoc mesenteroides is proposed. Neither the formation of theactive enzymes nor that of levan or dextran or the production oftransgenic plants is described. PCT/EP93/02110 discloses a method forproducing transgenic plants expressing the Isc gene of the levan sucrasefrom the gram-negative bacterium Erwinia amylovora. The plants produce ahigh-molecular, strongly branched levan. PCT/NL93/00279 describes thetransformation of plants with chimeric genes containing the sacB genefrom Bacillus subtilis or the ftf gene from Streptococcus mutans.Transgenic plants expressing the sacB gene produce a branched levan.Plants expressing the ftf gene synthesize high-molecular inulin; theyield, however, is so low that an economic utilization is out ofquestion. PCT/NL96/00012 discloses DNA sequences encoding enzymessynthesizing carbohydrate polymers as well as the production oftransgenic plants by means of these DNA sequences. The disclosedsequences are derived from Helianthus tuberosus. According toPCT/NL96/00012, the disclosed sequences may be used in order to modifythe fructan profile of petunia and potato, but also of Helianthustuberosus itself. When expressing the SST and the FFT gene in transgenicplants, it is possible to produce inulin. The average degree ofpolymerization of inulin, however, ranges between DP=6 and DP=10. Theproduction of high-molecular inulin is not possible by means of themethod described in PCT/NL96/00012. PCT/EP97/02195 describes a methodfor producing transgenic, inulin-producing plants by means of the ftfgene from Streptococcus mutans. The yield of high-molecular inulin islow, as is the case with the plants described in PCT/NL9300279. DE 19708 774.4 describes the production of short-chain inulin by means ofenzymes exhibiting fructosyl polymerase activity. The short-chain inulinmay be produced in transgenic plants. The yield of short-chain inuin ishigh and in potato it corresponds to the cellular content of sucrose.The production of long-chain inulin, however, is not described.

The synthesis of inulin in plants has been thoroughly examined (Pollock& Chafterton, Fructans, The Biochemistry of Plants Vol. 14 (1988),Academic Press, pp.109-140). However, the inulin occurring naturally inplants is short-chain fructan with a maximum degree of polymerization ofapproximately DP=35 (Pollock & Chatterton, 1988, loc.cit.). Synthesisand metabolism of fructans in plants are based on the activity of atleast three enzymes: a sucrose-dependent sucrose-fructosyl transferase(SST) forming the tri-saccharide kestose, a fructan-dependentfructan-fructosyl transferase (FFT) which transfers fructosyl residuesfrom fructan molecules with a minimum degree of polymerization of DP=3(kestose) to sucrose and higher fructans, and a fructan exohydrolase(FEH) which removes fructose residues from fructan molecules. It is notknown whether differences in the average molecular weight of the inulinin various plant species, for example about 2×10³ in the case of Alliumcepa and 5×10³ in the case of Helianthus tuberosus, are based on thedifferent properties of their SST, FFT or FEH.

For this reason it is not possible in view of the present knowledgerelating to the inulin synthesis in plants to identify suitable DNAsequences by means of which high-molecular inulin might be synthesizedin plants in economically interesting amounts.

Thus, the technical problem underlying the present invention is toprovide nucleic acid molecules and methods which allow for theproduction of genetically modified organisms, in particular plants,capable of forming long-chain inulin.

This problem is solved by the provision of the embodiments characterizedin the claims.

SUMMARY OF THE INVENTION

Therefore, the present invention relates to nucleic acid moleculesencoding proteins with the enzymatic activity of an FFT, selected fromthe group consisting of

(a) nucleic acid molecules encoding a protein comprising the amino acidsequence indicated under SEQ ID NO: 2 or SEQ ID NO: 4;

(b) nucleic acid molecules comprising the nucleotide sequence indicatedunder SEQ ID NO: 1 or SEQ ID NO: 3 or a corresponding ribonucleotidesequence;

(c) nucleic acid molecules which hybridize to a complementary strand ofthe nucleic acid molecules mentioned under (a) or (b) under stringentconditions; and

(d) nucleic acid molecules comprising a fragment of the nucleotidesequence of (a), (b) or (c).

In the context of the present invention a fructosyl transferase (FFT) isa protein capable of catalyzing the formation of β-2,1-glycosidic and/orβ-2,6-glycosidic bonds between fructose units. Thereby, a fructosylresidue to be transferred may be derived from 1-kestose or from afructan polymer. In connection with the present invention, ahigh-molecular fructan is a polymer the molecules of which contain anaverage number of more than 20, preferably more than 25 and even morepreferably at least 32 fructosyl residues. Furthermore, thehigh-molecular fructan is preferably a polymer the molecules of whichcontain on the average less than 3000, more preferably less than 300 andparticularly preferred less than 100 fructosyl residues. The fructosylresidues may be either glycosidically linked by β-2,1 bonds or by β-2,6bonds. In the case of inulin the residues are generally linked by β-2,1glycosidic bonds. To a low degree, also β-2,6-bonds may occur, inparticular by less than 5%, preferably by less than 3%, more preferablyby less than 1.5% and most preferably by less than 0.5%. The fructosylpolymer may carry at its end a glucose residue which is linked via theC-1 OH-group of the glucose and the C-2 OH-group of a fructosyl residue.In this case, a sucrose molecule is also contained in the fructosylpolymer.

Surprisingly, high amounts of high-molecular inulin are formed duringthe expression of the nucleic acid molecules of the invention intransformed plants. The inulin formed in the plants exhibits an averagedegree of polymerization of clearly more than DP=20. This was unexpectedsince a similar enzyme from Helianthus tuberosus is involved in thesynthesis of inulin with an average degree of polymerization of lessthan DP=20 in transgenic plants (PCT/NL96/00012).

The nucleic acid molecules of the invention may be DNA as well as RNAmolecules. Corresponding DNA molecules are for example genomic DNA orcDNA molecules. The nucleic acid molecules of the invention may beisolated from natural sources, preferably from artichoke, or they may besynthesized according to known methods. By means of conventionalmolecular-biological techniques it is possible (see e.g. Sambrook etal., 1989, Molecular Cloning, A Laboratory Manual, 2^(nd) edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) to introducevarious mutations into the nucleic acid molecules of the invention,which leads to the synthesis of proteins with probably modifiedbiological properties. In this respect, it is possible on the one handto produce deletion mutants, in which nucleic acid molecules areproduced by progressing deletions at the 5′ or 3′ end of the coding DNAsequence. These nucleic acid molecules lead to the synthesis, ofcorrespondingly shortened proteins. By means of such deletions at the 5′end of the nucleotide sequence it is for example possible to identifyamino acid sequences which are responsible for the translocation of theenzyme into the vacuole (transit peptides). This allows for the targetedproduction of enzymes which, due to the removal of the correspondingsequences, are no longer located within the vacuole but within thecytosol, or within other compartments due to the addition of othersignal sequences.

On the other hand, it is also conceivable to introduce point mutationsat positions in which a modification of the amino acid sequence forexample influences the enzyme activity or the regulation of the enzyme.In this manner e.g. mutants may be produced which exhibit a modifiedK_(m) value or which are no longer subject to the regulation mechanismsoccurring in the cell, such as allosteric regulation or covalentmodification.

Furthermore, mutants may be produced which exhibit a modified substrateor product specificity. Furthermore, mutants with a modifiedactivity-temperature-profile may be produced.

For recombinant DNA manipulation in prokaryotic cells, the nucleic acidmolecules of the invention or parts of these molecules may be insertedinto plasmids which allow for a mutagenesis or a sequence modificationby recombination of DNA sequences. By means of standard techniques (cf.Sambrook et al., 1989, Molecular Cloning: A laboratory manual, 2^(nd)edition, Cold Spring Harbor Laboratory Press, N.Y., USA) base exchangesmay be carried out or natural or synthetic sequences may be added. Inorder to link the DNA fragments to each other, adapters or linkers maybe connected with the fragments. Furthermore, manipulations may be usedwhich provide suitable restriction sites or which remove superfluous DNAor restriction sites. If use can be made of insertions, deletions orsubstitutions, in vitro mutagenesis, primer repair, restriction orligation may be used. As analyzing method, use is usually made ofsequence analysis, restriction analysis or furtherbiochemico-molecular-biological methods.

In the context of the present invention the term “hybridization” meanshybridization under conventional conditions, preferably under stringentconditions, as described for example in Sambrook et al., MolecularCloning, A Laboratory Manual, 2^(nd) edition (1989), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. An example for stringenthybridization conditions is a hybridization in 50% formamide, 5×SSC,5×Denhardt's solution, 40 mM sodium phosphate pH 6.8; 0.5% (w/v) BSA, 1%(w/v) SDS, 0.1 mg/ml herring sperm DNA at 42° C. An example forconventional non-stringent hybridization conditions is a hybridizationunder the above-described conditions in which, however, 30% formamide isused instead of 50%. Washing conditions in the case of stringentconditions are preferably 0.5×SSC/0.5% SDS at 60° C. and in the case ofnon-stringent conditions preferably 2×SSC/0.5% SDS at 56° C.

Nucleic acid molecules which hybridize to the molecules of the inventioncan e.g. be isolated from genomic or from cDNA libraries produced fromcorresponding organisms, such as artichoke.

Such nucleic acid molecules may be identified and isolated by using themolecules of the invention or parts of these molecules or, as the casemay be, the reverse complements of these molecules, e.g. byhybridization according to standard techniques (see e.g. Sambrook etal., 1989, Molecular Cloning, A Laboratory Manual, 2^(nd) edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). As ahybridization probe e.g. nucleic acid molecules may be used whichexhibit exactly or basically the nucleotide sequence indicated under SEQID No. 1 or SEQ ID No. 3 or parts thereof. The fragments used ashybridization probe may also be synthetic fragments produced by means ofthe usual synthesis techniques and the sequence of which is basicallysimilar to that of a nucleic acid molecule of the invention.

The molecules hybridizing to the nucleic acid molecules of the inventionalso comprise fragments, derivatives and allelic variants of theabove-described nucleic acid molecules encoding a protein of theinvention. “Fragments” are supposed to be parts of the nucleic acidmolecules which are long enough in order to encode a protein of theinvention. In this context, the term “derivative” means that thesequences of these molecules differ from the sequences of theabove-described nucleic acid molecules at one or more positions.However, they exhibit a high degree of homology to these sequences.Homology means a sequence identity of at least 40%, in particular anidentity of at least 60%, preferably of more than 80% and mostpreferably of more than 90%. The proteins encoded by these nucleic acidmolecules exhibit a sequence identity to the amino acid sequenceindicated under SEQ ID No. 2 of at least 80%, preferably 85% andparticularly preferred of more than 90%, more preferred of more than95%, even more preferred of more than 97% and most preferred of morethan 99%. The deviations from the above-described nucleic acid moleculesmay, for example, result from deletion, substitution, insertion and/orrecombination.

The nucleic acid molecules which are homologous to the above-describedmolecules and represent derivatives of these molecules, are usuallyvariations of these molecules representing modifications with the samebiological function. These may be naturally occurring variations, forexample sequences from other organisms, or mutations, whereby thesemutations may have occurred naturally or they may have been introducedby means of targeted mutagenesis. Furthermore, the variations may besynthetically produced sequences. The allelic variants may either benaturally occurring variants or synthetically or recombinantly producedvariants. The proteins encoded by the various variants of the nucleicacid molecules of the invention exhibit certain common characteristicssuch as the enzyme activity, molecular weight, immunological reactivityor conformation or physical properties such as the mobility in gelelectrophoresis, chromatographic characteristics, sedimentationcoefficients, solubility, spectroscopic properties, stability,pH-optimum, temperature-optimum etc.

In a preferred embodiment the nucleic acid sequences of the inventionare derived from artichoke (Cynara scolymus).

The invention further relates to vectors containing the nucleic acidmolecules of the invention. These are preferably plasmids, cosmids,viruses, bacteriophages and other vectors common in gene technology.

Within the vector of the invention the nucleic acid molecule of theinvention is preferably operably linked to regulatory elements whichensure the transcription and synthesis of a translatable RNA inprokaryotic and/or eukaryotic cells.

The expression vectors of the invention allow for the production oflong-chain inulin in various host organisms, in particular inprokaryotic or eukaryotic cells such as bacteria, fungi, algae, animalcells and preferably plant cells and plants. Preferred host organismsare in particular yeasts such as e.g. S. cerevisiae, and lactic acidbacteria such as Streptococcus thermophilus, Lactobacillus bulgaricus,Streptococcus lactis, S. cremoris, Lactobacillus acidophilus andLeuconostoc cremoris. The encoded enzymes may probably also be usedoutside of the host organisms for the production of long-chain inulin.Plant cells are particularly preferred.

A survey concerning various expression systems may be found e.g. inMethods in Enzymology 153 (1987), 385-516, in Bitter et al. (Methods inEnzymology 153 (1987), 516-544), Sawers et al., Applied Microbiology andBiotechnology 46 (1996), 1-9, Billmann-Jacobe, Current Opinion inBiotechnology 7 (1996), 500-504, Hockney, Trends in Biotechnology 12(1994), 456-463, and Griffiths et al., Methods in Molecular Biology 75(1997), 427-440. Expression systems for yeast have been described inHensing et al., Antonie van Leuwenhoek 67 (1995), 261-279, Bussineau etal., Developments in Biological Standardization 83 (1994), 13-19,Gellissen et al., Antonie van Leuwenhoek 62 (1992), 79-93, Fleer,Current Opinion in Biotechnology 3 (1992), 486-496, Vedvick, CurrentOpinion in Biotechnology 2 (1991), 742-745, and in Buckholz,Bio/Technology 9 (1991), 1067-1072. Expression vectors have beendescribed to a great extent in the prior art. Apart from a selectionmarker gene and a replication origin ensuring replication in theselected host, they usually contain a bacterial or viral promoter and inmost cases a termination signal for transcription. There is at least onerestriction site or one polylinker between the promoter and thetermination signal which allows to insert a coding DNA sequence. If itis active in the selected host organism, the DNA sequence naturallycontrolling the transcription of the corresponding gene may be used aspromoter sequence. This sequence may also be exchanged with otherpromoter sequences. Use may also be made of promoters which lead to aconstitutive expression of the gene as well as of inducible promotersallowing for a targeted regulation of the expression of the downstreamgene. Bacterial and viral promoter sequences with these properties havebeen extensively described in the prior art. Regulatory sequences forthe expression in microorganisms (such as E. coli, S. cerevisiae) havebeen sufficiently described in the prior art. Promoters which allow fora particularly strong expression of the downstream gene are e.g. the T7promoter (Studier et al., Methods in Enzymology 185 (1990), 60-89),lacuv5, trp, trp-lacUV5 (DeBoer et al., in Rodriguez and Chamberlin(eds.), Promoters, Structure and Function; Praeger, New York (1982),462-481; DeBoer et al., Proc. Natl. Acad. Sci. USA (1983), 21-25), λp1,rac (Boros et al., Gene 42 (1986), 97-100). Usually, the amounts ofprotein are highest from the middle towards the end of the logarithmicphase of the microorganisms' growth cycle. For this reason, induciblepromoters are preferably used for the synthesis of proteins. Thesefrequently lead to higher protein yields than constitutive promoters.The use of strongly constitutive promoters often leads, via thepermanent transcription and translation of the cloned gene, to the lossof energy for other essential cell functions, which slows down thegrowth of the cell (Bernard R. Glick, Jack J. Pasternak, MolekulareBiotechnologie (1995), Spektrum Akademischer Verlag GmbH, HeidelbergBerlin Oxford, p. 342). Thus, in order to reach an optimum amount ofprotein a two-stage process is often used. At first, host cells arecultivated under optimum conditions until a relatively high cell densityis achieved. In the second stage, transcription is induced depending onthe kind of promoter used. In this context, a tac-promoter inducible bylactose or IPTG (=isopropyl-β-D-thiogalacto-pyranosid) is particularlysuitable (deBoer et al., Proc. Natl. Acad. Sci. USA 80 (1983), 21-25).Termination signals for the transcription are also described in theprior art.

The transformation of the host cell with the correspondingprotein-encoding DNA may generally be carried out by means of standardtechniques, such as described by Sambrook et al. (Molecular Cloning: ALaboratory Course Manual, 2^(nd) edition (1989), Cold Spring HarborPress, New York). The cultivation of the host cell takes place innutrient media which correspond to the respective requirements of thehost cells used, particularly considering the pH value, temperature,salt concentration, airing, antibiotics, vitamins, trace elements etc.

The purification of the enzyme produced by the host cells may be carriedout by means of conventional purification techniques such asprecipitation, ion exchange chromatography, affinity chromatography, gelfiltration, HPLC reverse phase chromatography etc.

By modifying the DNA expressed in the host cells, a polypeptide may beproduced in the host cell which can easier be isolated from the culturemedium due to certain properties. Thus, there is the possibility ofexpressing the protein to be expressed as a fusion protein with afurther polypeptide sequence, the specific binding properties of whichallow for the isolation of the fusion protein via affinitychromatography (e.g. Hopp et al., Bio/Technology 6 (1988), 1204-1210;Sassenfeld, Trends Biotechnol. 8 (1990), 88-93).

For expression in plant cells, regulatory elements of the patatin B33promoter are preferred. Other preferred promoters are the 35S CaMVpromoter and the promoter of the alcohol dehydrogenase gene fromSaccharomyces cerevisiae.

The vectors of the invention may possess further functional units whichstabilize the vector within a host organism, e.g. a bacterialreplication origin or the 2-micron-DNA for stabilization inSaccharomyces cerevisiae. Furthermore, they may contain left and rightborder sequences of agrobacterial T-DNA, thus enabling a stableintegration into the genome of plants.

The vectors of the invention may further contain functional terminators,such as the terminator of the octopin synthase gene from Agrobacteria.

In another embodiment the nucleic acid molecule of the invention islinked to a nucleic acid molecule within the vector of the invention,said nucleic acid molecule encoding a functional signal sequence inorder to direct the enzyme to various cell compartments. Thismodification may for example consist in an addition of an N-terminalsignal sequence for the secretion into the apoplast of higher plants;however, any other modification leading to the fusion of a signalsequence to the encoded FFT is also a subject matter of the invention.The nucleic acid molecule contained in the vector of the invention mayin particular contain a sequence encoding an amino acid sequence causingsecretion. In this context, use is preferably made of the signal peptideof the α-CGTase from Klebsiella oxytoca M5A1 (Fiedler et al., J. Mol.Biol. 256 (1996), 279-291) or of a signal peptide as it is encoded bythe nucleotides 11529-11618 of the sequence with the gene bank accessionnumber X 86014.

In a particularly preferred embodiment the invention relates to plasmidsp35-csFFT and p33-csFFT, the construction of which is described in theexamples (FIG. 2 and 4).

In a further embodiment the invention relates to host cells, whichtransiently or stably contain the nucleic acid molecules or vectors ofthe invention or are derived from such cells. In this context, a hostcell is an organism capable of taking up recombined DNA in vitro and, ifapplicable, of synthesizing the proteins encoded by the nucleic acidmolecules of the invention. The host cells may be prokaryotic as well aseukaryotic cells. They may in particular be microorganisms. In thecontext of the present invention, these are all bacteria and protists(such as fungi, in particular yeasts and algae) as they are defined e.g.in Schlegel “Allgemeine Mikrobiologie” (Georg Thieme Verlag (1985),1-2). In connection with prokaryotic host organisms it should be notedthat the positive influence of inulin on the growth of certainmicroorganisms, such as Bifido bacteria, of the human intestinal tracthas successfully been shown. Bifido bacteria have been ascribed ahealthy effect (see e.g. Gibson et al., Int. Sugar J. 96 (1994),381-386; Roberfroid et al., J. of Nutrition 128 (1998), 11-19). Atumor-inhibiting effect has also been discussed (see e.g. Reddy et al,Carcinogenesis 18 (1997), 1371-1374; Singh et al., Carcinogenesis 18(1997), 833-841). For this reason, the host cells of the invention suchas yeast (bread) or lactic acid bacteria (yogurt, butter-milk etc.) aresuitable for use in the food processing industry.

In a particularly preferred embodiment a host cell of the inventionadditionally contains a gene encoding a sucrose-dependent sucrosefructosyl transferase (SST). Such sequences were, for example, isolatedfrom artichoke (German patent application DE-A1 197 08 774), Cichoriumintibus (de Halleux et al., Plant Physiol. 113 (1997), 1003-1013),Helianthus tuberosus (WO 96/21023) and Allium cepa (Vijn et al., PlantPhysiol. 117 (1998), 1507-1513).

The invention in particular relates to transgenic plant cellstransformed with a nucleic acid molecule of the invention or containingthe vector systems of the invention or derivatives or parts thereof.These are capable of synthesizing enzymes for the production oflong-chain inulin due to the introduction of the vector systems of theinvention, derivatives or parts of the vector system. The cells of theinvention are preferably characterized in that the introduced nucleicacid molecule of the invention is either heterologous with respect tothe transformed cell, i.e. it does not naturally occur in these cells oris localized at a different position within the genome than therespective naturally occurring sequence. Moreover, such a transgenicplant cell of the invention preferably contains a DNA sequence encodinga SST.

The present invention further relates to proteins encoded by the nucleicacid molecules of the invention, as well as to methods for theirproduction wherein the host cell of the invention is cultivated underconditions which allow for the synthesis of the protein. The protein issubsequently isolated from the cultivated cells and/or from the culturemedium. The invention further relates to an FFT obtainable from the hostcell of the invention or by a method of the invention.

The invention further relates to nucleic acid molecules whichspecifically hybridize to a nucleic acid molecule of the invention, to amolecule complementary thereto or to a part of such molecules. These arepreferably oligonucleotides with a length of at least 10, in particularof at least 15 and particularly preferred of at least 50 nucleotides.The oligonucleotides of the invention may for example be used as primersfor a PCR reaction. They may also be components of antisense constructsor of DNA molecules encoding suitable ribozymes.

The present invention also relates to a method for the production oftransgenic plant cells, plant tissue and plants comprising theintroduction of a nucleic acid molecule or vector of the invention intoplant cells, plant tissue and plants. By providing the nucleic acidmolecules of the invention it is possible by means of recombinant DNAtechniques to produce long-chain inulin in various organisms, inparticular in plants, as it was so far impossible by means ofconventional, e.g. breeding methods. By increasing the activity of theFFT of the invention, for example by overexpressing the nucleic acidmolecules of the invention, or by providing mutants that are no longersubject to cell-specific regulation mechanisms and/or exhibit distincttemperature dependencies with respect to their activity, it is possibleto increase the yield of plants correspondingly modified by means ofrecombinant DNA techniques.

Thus, it is possible to express the nucleic acid molecules of theinvention in plant cells in order to increase the activity of thecorresponding FFT, or to introduce it into cells that do not normallyexpress this enzyme. It is furthermore possible to modify the nucleicacid molecules of the invention according to methods known to theskilled person, in order to obtain the FFTs of the invention that are nolonger subject to cell-specific regulation mechanisms or which exhibitmodified temperature-dependencies, substrate or product specificities.

For this purpose, the skilled person may utilize various planttransformation systems. Thus, the use of T-DNA for transforming plantcells has been intensely examined and described in EP-A-120 516;Hoekema: The Binary Plant Vector System, Offsetdrukkerij Kanters B. V.,Alblasserdam (1985), Chapter V, Fraley, Crit. Rev. Plant. Sci., 4, 1-46and An, EMBO J. 4 (1985), 277-287.

For transferring the DNA into the plant cells, plant explants maysuitably be co-cultivated with Agrobacterium tumefaciens orAgrobacterium rhizogenes. From the infected plant material (e.g. piecesof leaves, stem segments, roots, but also protoplasts orsuspension-cultivated plant cells) whole plants may then be regeneratedin a suitable medium which may contain antibiotics or biozides for theselection of transformed cells. The plants obtained in such a way maythen be examined as to whether the introduced DNA is present or not.Other possibilities in order to introduce foreign DNA by using thebiolistic method or by transforming protoplasts are known to the skilledperson (cf. e.g. Willmitzer, L., 1993 Transgenic plants. In:Biotechnology, A Multi-Volume Comprehensive Treatise (H. J. Rehm, G.Reed, A. Pühler, P. Stadler, editors), Vol. 2, 627-659, VCH Weinheim-NewYork-Basel-Cambridge).

Alternative Systems for the transformation of monocotyledonous plantsare the transformation by means of the biolistic approach, theelectrically or chemically induced uptake of DNA into protoplasts, theelectroporation of partially permeabilized cells, the macro-injection ofDNA into inflorescences, the micro-injection of DNA into microspores andpro-embryos by means of swelling (see e.g. Lusardi, Plant J. 5 (1994),571-582; Paszkowski, Biotechnology 24 (1992), 387-392). Whereas thetransformation of dicotyledonous plants by Ti-plasmid-vector systems bymeans of Agrobacterium tumefaciens is a well-established method, morerecent studies indicate that the transformation with vectors based onAgrobacterium can also be used in the case of monocotyledonous plants(Chan et al., Plant Mol. Biol. 22 (1993), 491-506; Hiei et al., Plant J.6 (1994), 271-282; Bytebier et al., Proc. Natl. Acad. Sci. USA 84(1987), 5345-5349; Raineri et al., Bio/Technology 8 (1990), 33-38; Gouldet al., Plant Physiol. 95 (1991), 426-434; Mooney et al., Plant, CellTiss. & Org. Cult. 25 (1991), 209-218; Li et al., Plant Mol. Biol. 20(1992), 1037-1048).

Three of the above-mentioned transformation systems have in the pastbeen established for various types of cereals: electroporation of planttissue, transformation of protoplasts and the DNA-transfer byparticle-bombardment into regenerable tissue and cells (review given in:Jahne et al., Euphytica 85 (1995), 35-44). In the correspondingliterature the transformation of wheat is described in various ways(reviewed in Maheshwari et al., Critical Reviews in Plant Science 14 (2)(1995), 149-178).

When expressing the nucleic acid molecules of the invention in plants itis in principle possible that the synthesized protein may be localizedwithin any desired compartment of the plant cell. In order to achievethe localization in a particular compartment the sequence ensuring thelocalization within the vacuole must be deleted and the remaining codingregion has, optionally, to be linked to DNA sequences which ensure thelocalization within the respective compartment. Such sequences are knownin the art (see for example Braun, EMBO J. 11 (1992), 3219-3227; Wolter,Proc. Natl. Acad. Sci. USA 85 (1988), 846-850; Sonnewald, Plant J. 1(1991), 95-106; Rocha-Sosa, EMBO J. 8 (1989), 23-29).

The present invention also relates to transgenic plant cells, planttissue and plants which were transformed with one or several of thenucleic acid molecules of the invention, as well as to transgenic plantcells derived from cells transformed in such a way. Such cells containone or several of the nucleic acid molecules of the invention, wherebythis/these is/are preferably linked to regulatory DNA elements thatensure transcription in plant cells, in particular with a promoter. Suchcells differ from naturally occurring plant cells in that they containat least one nucleic acid molecule of the invention which does notnaturally occur in these cells or in that such a molecule is integratedat a position within the genome of the cell where it does not naturallyoccur, i.e. in another genomic environment. Since 1-kestose is thenatural substrate of FFT and is itself formed in the reaction of asucrose-dependent sucrose fructosyl transferase (SST) with the sucrose,it is particularly advantageous and probably necessary to provide an SSTapart from the nucleic acid molecule, vector or FFT of the invention.Thus, in a preferred embodiment the present invention relates totransgenic plant cells, plant tissue or plants which additionallycontain a gene encoding a sucrose-dependent sucrose fructosyltransferase (SST). These may for example be plants or plant cells whichalready naturally express an SST such as chicory, Helianthus tuberosus,or dahlia or plants into which an SST-encoding DNA sequence wasintroduced by means of recombinant DNA techniques. Said sequence mayhave been introduced independently or simultaneously with a nucleic acidmolecule or vector of the invention.

The transgenic plant cells and plant tissues can be regenerated to wholeplants by means of techniques known to the skilled person. The plantsobtainable by regenerating the transgenic plant cells of the inventionare also a subject matter of the present invention. A further subjectmatter of the invention are plants which contain the above-describedtransgenic plant cells. The transgenic plant cells may in principle beany desired kind of plant species, i.e. monocotyledonous as well asdicotyledonous plants. They are preferably useful plants, in particularsucrose-containing plants such as rice, maize, sugar beet, sugar cane orpotato, vegetable plants (e.g. tomato, carrot, leek, chicory etc.),feeding or pasture grass, sweet potato, wheat, barley, rape or soy bean.

The invention also relates to propagation material and harvest productsof the plants of the invention such a fruits, seeds, tubers, rootstocks,seedlings, cutting, calli, cell cultures etc.

A further subject matter of the invention is the long-chain inulinobtainable from the host cells of the invention, in particular fromtransgenic plant cells, plant tissues, plants as well as from thepropagation material and from the harvest products.

In another embodiment the invention relates to methods for producinglong-chain inulin comprising:

(a) cultivating a host cell, particularly a plant cell, plant tissue ora plant of the invention, under conditions which allow for theproduction of FFT and the conversion of 1-kestose, optionally suppliedfrom the outside, or of an equivalent substrate into long-chain inulin;and

(b) recovering the thus produced inulin from the cultivated host cells,in particular plant cells, tissues or plants, or from the medium.

In a further embodiment the invention relates to a method for theproduction of long-chain inulin comprising:

(a) bringing 1-kestose or an equivalent substrate into contact with anFFT of the invention under conditions which allow for the conversioninto long-chain inulin; and

(b) recovering the thus produced inulin.

The recovering of the inulin from various sources, in particular fromplant tissue, has for example been described in Gibson et al., Int.Sugar J. 96 (1994), 381-386; Baxa, Czech J. Food Sci. 16 (1998), 72-76;EP-A-787 745; De Leenheer, Carbohydr. Org. Raw Mater. III, Workshop(1996), Meeting Date 1994, 67-92, Verlag VCH Weinheim, Germany andRussian patent RU 2001621 C1.

The present invention further relates to an in vitro method forproducing long-chain inulin by using the substrate sucrose and an enzymecombination from an SST and an FFT of the invention. In a furtherembodiment the present invention relates to an in vitro method forproducing inulin by using a mixture containing fructosyl oligomers andan FFT of the invention. In this context, a fructosyl oligomer is anoligomer consisting of fructose units with a DP of approximately 2 to 7which may exhibit a glucose residue at its end. When carrying out themethod of the invention, recombinantly produced proteins are preferablyused. In the context of the present invention these are proteins whichwere produced by introducing the respective protein-encoding DNAsequence into a host cell and expressing it there. The protein maysubsequently be recovered from the host cell and/or from the culturemedium. The host cell is preferably a host cell of the invention asdefined above. In a preferred embodiment of the method of the inventionenzymes are used which were recombinantly produced and secreted into theculture medium by the host cell, so that it is not necessary to disruptthe cells or to further purify the protein since the secreted proteinmay be obtained from the supernatant. In order to remove the residues ofthe culture medium, conventional processing techniques may be used suchas dialysis, reverse osmosis, chromatographic methods etc. The sameholds true for concentrating the protein secreted in the culture medium.The secretion of proteins by microorganisms is normally mediated byN-terminal signal peptides (signal sequence, leader peptide). Proteinswith this signal sequence may penetrate the cell membrane of themicroorganism. A secretion of proteins may be achieved by linking theDNA sequence encoding this signal peptide to the correspondingenzyme-encoding region. Use is preferably made of the signal peptide ofthe α-CGTase from Klebsiella oxytoca M5A1 (Fiedler et al., J. Mol. Biol.256 (1996), 279-291) or of a signal peptide as it is encoded by thenucleotides 11529-11618 of the sequence deposited in the gene bank withthe accession number X86014.

The enzymes used in the method of the invention may alternatively beproduced not by using microorganisms but by means of an in vitrotranscription and translation system which leads to the expression ofthe proteins. In a particularly preferred embodiment of the inventionthe FFT is produced from the protoplasts of the leave tissue in plants.

The invention further relates to inulin which may be formed from a hostcell, in particular a plant cell, plant tissue or a plant of theinvention or from the propagation material or the harvest product of theplants and plants cells of the invention or which is obtained by one ofthe above-described methods of the invention. This inuslin maypreferably be used in order to produce surfactants for increasing theviscosity in aqueous system, as a suspending agent, for speeding upsedimentation, for complexing or for binding water.

These or other embodiments have been disclosed and are evident to theskilled person. They are comprised by the description and the examplesof the present invention. Further literature that relates to one of theabove-mentioned methods, means or uses and that can be applied in thesense of the present invention, may be taken from the prior art, e.g.from public libraries or by utilizing electronic means. Public databases serve this purpose, as e.g. “Medline” which may be accessed viaInternet. Further data bases and addresses are known to the personskilled in the art and may be taken from the Internet. A survey ofsources and information regarding biotechnology patents or patentapplications can be found in Berks, TIBTECH 12 (1994), 352-364.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the HPLC analysis of a complete protoplast extract. Theprotoplasts were transformed with various vectors: A: transformation wascarried out with the vector pA7 that does not contain a coding regionfused to the CaMV 35S promoter. B: transformation took place with thevector pA7-csFFT which contains the coding region of thefructan:fructan-fructosyl transferase from artichoke fused to the CaMV35S promoter. C: transformation was carried out with the vectorpA7-htFFT which contains the coding region of the fructan:fructanfructosyl transferase from Helianthus tuberosus as a fusion to the CaMV35S promoter. Before analysis, the complete protoplast extracts wereincubated in a mixture of fructosyl oligomers for 12 h each. Analysiswas carried out as described in Example 1.

FIG. 2 shows the construction of the plasmid p35-csFFT

FIG. 3 shows the HPLC analysis of transgenic plants which weretransformed with the construct p35-csFFT. The analysis shows thatlong-chain inulin molecules were formed in transgenic plants whichexpress an SST as well as an FFT from artichoke (35S-SST/FFT 22/19).

FIG. 4 shows the construction of the plasmid p33-csFFT

FIG. 5 shows the HPLC analysis of transgenic plants which weretransformed with the construct p33-csFFT. The analysis shows thatlong-chain inulin molecules were formed in transgenic plants whichexpress an SST as well as an FFT from artichoke (B33-SST/FFT 47).

DETAILED DESCRIPTION OF THE INVENTION

The Examples illustrate the invention.

EXAMPLE 1 Identification, Isolation and Characterization of a cDNAEncoding a Fructosyl Transferase from Artichoke (Cynara scolymus)

Total RNA was isolated from the receptacles of artichoke (Sambrook etal., 1989, Molecular Cloning, A Laboratory Manual, 2^(nd) edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA). Poly(A)+mRNA was isolated by means of the mRNA isolation system PolyATract(Promega Corporation, Madison, Wis., USA). Complementary DNA (cDNA) wasproduced from 5 μg of this RNA by means of the ZAP-cDNA synthesis kit ofStratagene (Heidelberg) according to the manufacturer's instructions,and 2×10⁶ independent recombinant phage clones were obtained. Theamplified cDNA library was screened according to standard methods underlow stringency conditions by means of the ³²P-labeled DNA fragmentcorresponding to the cDNA of the SST from artichoke (Not I-fragment ofthe plasmid pCy21 as described in DE 197 08 774.4). The sequence of theSST from artichoke has been described in DE 197 08 774.4. Positiveclones were screened by means of the SST probe under high stringency.Clones which reacted positively during this screening were abandonedsince they were evidently SST cDNA. From the residual clones the cDNAinsert was isolated by cleaving the plasmid DNA isolated in standardroutines by means of the restriction enzyme NotI and was cloned into thevector pA7. The sticky ends of the NotI fragment were filled in by meansof the T4 polymerase. Subsequently, the fragment was ligated into theSmaI site of pA7. The vector pA7 is a derivative of pUC18(Yanish-Perron, Gene 33 (1985), 103-119) which contains an insert of the35S promoter of the Cauliflower-Mosaic virus (nucleotide 7146 to 7464according to Gardner, Nucleic Acids Res. 9 (1981), 2871-2888) betweenthe EcoRI and the SacI site of the polylinker. Apart from the 35Spromoter, pA7 contains the polyadenylation signal of gene 3 of the T-DNAof the Ti plasmid pTi ACH 5 (Gielen, EMBO J. 3 (1984), 835-846),nucleotides 11749 to 11939, which was isolated as a Pvu II-Hind IIIfragment from the plasmid pAGV 40 (Herrera-Estrella, Nature 303 (1983),209-213) and cloned between the SphI and the Hind III site of thepolylinker after adding Sph I linkers to the Pvu II site.

By means of the pA7 derivatives which contained a cDNA from artichoke,tobacco protoplasts were transformed according to the method of Negrutiu(Plant Mol. Biol. 8, (1987), 363-373). The transformed protoplasts werecultivated in K3 medium (Nagy and Maliga, Z. Pflanzenphysiologie 78(1976), 453-455) at 25° C. for two days in the dark. Subsequently, thecell extracts were obtained by repeated freezing and thawing. Theextracts were incubated with oligofructans (67.5% 1-kestose, 28.4%nystose, 3.6% fructosyl nystose, 0.5% sucrose) for 12 h at 28° C. andsubsequently analyzed by HPLC. The HPLC analysis was carried out with aCarboPac PA 100 anionic exchange column, which was connected to a DionexDX-300 gradient chromatography system (Dionex, Sunnyvale, Calif., USA).Sugar monomers, oligomers and polymers were detected by means ofpulsamperometric detection. The detector adjustment for this purposewas: T₁=0.48s; T₂=0.12s; T₃=0.12s; E₁=0.05V; E₂=0.65V; E₃=−0.95V;sensibility=0.1 μC; integration =0.28-0.48s; flow medium A=0. 15 M NaOH;flow medium B=1 M NaAc in 0.15 M NaOH; gradient: 10 min 100% A; 2 minlinear increase from 0% B to 100% B; 2 min 100% B; 2 min linear increasefrom=0% A to 100% A; 5 min A. The samples were desalinated and filtered(microcon 10, amicon, Beverly, USA) before application. The flow speedwas 1 ml min⁻¹. In a few extracts, high-molecular inulin could be found(cf. FIG. 1).

EXAMPLE 2 Sequence Analysis of the cDNA Insert of the Plasmid pCy3

A cDNA insert from a pA7 derivative (pCy3) which had mediated thesynthesis of high-molecular inulin in the protoplast assay was sequencesby means of the didesoxynucleotide technique (Sanger, Proc. Natl. Acad.Sci. USA 74 (1977), 5463-5467). The insert of the clone pCy3 is a DNAwith a length of 2073 bp. The nucleotide sequence is indicated under SEQID No. 1. The corresponding amino acid sequence is indicated under SEQID No. 2. SEQ ID No. 3 is a variant of SEQ ID No. 1 which encodes thesame protein as that encoded by SEQ ID No. 1.

A sequence analysis and a comparison with already published sequenceshas shown that the sequence indicated under SEQ ID No. 1 is novel andcomprises a coding region exhibiting homologies to FFTs from otherorganisms.

EXAMPLE 3 Synthesis of the Plasmid p35-csFFT and Integration of thePlasmid into the Potato Genome

The plasmid p35-csFFT (FIG. 2) contains three fragments A, B and Cwithin the binary vector pBin19 (Bevan, Nucl. Acids Res. 12 (1984),8711, modified according to Becker, Nucleic Acids Res. 18 (1990), 203).

The fragment A contains the 35S promoter of the Cauliflower-Mosaic virus(CaMV). It contains the nucleotides 7146 to 7464 (Gardner, Nucleic AcidsRes. 9 (1981), 2871-2888) as an insert between the EcoRI and the SacIsite of the polylinker of pBin19-Hyg.

The fragment B contains the nucleotides 1 to 2073 of the sequence SEQ IDNo. 1. The fragment B was obtained as a Not I fragment from the vectorpBK-CMV into which it was inserted at the EcoRI site via an EcoRI/Not Ilinker sequence. The fragment C contains the polyadenylation signal ofthe gene 3 of the T-DNA of the Ti plasmid pTi ACH 5 (Gielen, EMBO J. 3(1984), 835-846), nucleotide 11749 to 11939, which was isolated as a PvuII-Hind III fragment from the plasmid pAGV 40 (Herrera-Estrella, Nature303 (1983), 209-213) and cloned between the SphI and the Hind III siteof the polylinker of pBin19-Hyg after adding Sph I linkers to the Pvu IIsite.

The plasmid p35-csSST was introduced into Agrobacteria (Höfgen andWillmitzer, Nucleic Acids Res. 16 (1988), 9877) and subsequentlyintroduced into potato plants via the Agrobacterium-mediated genetransfer according to the above-described standard techniques. Saidpotato plants were transformed with a DNA sequence encoding an SST fromartichoke (see German patent application DE-A1 197 08 774) and whichexpress these sequences under the control of the 35S promoter. Intactplants were regenerated from transformed cells. Extracts were obtainedfrom the leaves of regenerated plants and examined with respect to thepresence of fructosyl polymers. The analysis was carried out asdescribed in Example 1. The analysis of leaves from a range of plantstransformed with this vector system unambiguously proved the occurrenceof high-molecular inulin, which results from the expression of the FFTgene from artichoke contained in p35-csFFT (cf. FIG. 3).

TABLE I Analysis of inulin content of transgenic potato tubersexpressing an artichoke SST and FFT gene fructan content μmol fructose/average degree of polymerization Plant No. g fresh weight(fructose/glucose ration 35-SST/FFT 22/26 30.81 21 (20/1) 35-SST/FFT36/17 27.34 20 (19/1)

EXAMPLE 4 Production of the Plasmid p33-csFFT and Integration of thePlasmid into the Potato Genome

The plasmid p33-csFFT (FIG. 4) is identical with the plasmid p35-csFFT,with the exception that the fragment A contains the B33 promoter of thepatatin gene b33 from potato instead of the 35S promoter of CaMV. Itcontains a Dral fragment (position −1512 to position +14) of the patatingene b33 (Rocha-Sosa, EMBO J. 8 (1989), 23-29), which was insertedbetween the EcoRI and the SacI site of the polylinker of pBin19-Hyg. Theplasmid p33-csFFT has a size of approximately 14 kb. The plasmidp33-csSST was introduced into potato plants via theAgrobacterium-mediated gene transfer, as described in Example 3. Saidpotato plants were transformed with a DNA sequence encoding an SST fromartichoke (see German patent application E-A1 197 08 774) and whichexpressed these sequences under the control of the B33 promoter. Intactplants were regenerated from transformed cells. The analysis of tubersfrom a range of plants transformed with this vector system unambiguouslyproved the occurrence of high-molecular inulin, which results from theexpression of the FFT gene from artichoke contained in p33-csFFT (cf.FIG. 5).

4 1 2073 DNA Cynara scolymus CDS (21)..(1871) 1 ttacctcatt tccatcaaccatg aga acg act gaa ccc caa act gac ctt gag 53 Met Arg Thr Thr Glu ProGln Thr Asp Leu Glu 1 5 10 cat gca ccc aac cac act cca cta ctg gac cacccc gaa cca cca ccg 101 His Ala Pro Asn His Thr Pro Leu Leu Asp His ProGlu Pro Pro Pro 15 20 25 gcc gcc gtg aga aac cgg ttg ttg att agg gtt tcgtcc agt atc aca 149 Ala Ala Val Arg Asn Arg Leu Leu Ile Arg Val Ser SerSer Ile Thr 30 35 40 ttg gtc tct ctg ttt ttt gtt tca gca ttc cta ctc attctc ctg tac 197 Leu Val Ser Leu Phe Phe Val Ser Ala Phe Leu Leu Ile LeuLeu Tyr 45 50 55 caa cac gat tcc act tac acc gat gat aat tca gca ccg tcggaa agt 245 Gln His Asp Ser Thr Tyr Thr Asp Asp Asn Ser Ala Pro Ser GluSer 60 65 70 75 tct tcc cag cag ccc tcc gct gcc gat cgc ctg aga tgg gagaga aca 293 Ser Ser Gln Gln Pro Ser Ala Ala Asp Arg Leu Arg Trp Glu ArgThr 80 85 90 gct ttt cat ttc cag ccc gcc aaa aat ttc att tat gat ccc aacggt 341 Ala Phe His Phe Gln Pro Ala Lys Asn Phe Ile Tyr Asp Pro Asn Gly95 100 105 cca ttg ttc cat atg ggt tgg tac cat ctt ttc tac caa tac aacccg 389 Pro Leu Phe His Met Gly Trp Tyr His Leu Phe Tyr Gln Tyr Asn Pro110 115 120 tac gca ccg ttt tgg ggc aac atg aca tgg ggt cac gcc gtg tccaaa 437 Tyr Ala Pro Phe Trp Gly Asn Met Thr Trp Gly His Ala Val Ser Lys125 130 135 gac atg atc aac tgg ttc gag ctt ccg atc gcc ttg gcc cca accgaa 485 Asp Met Ile Asn Trp Phe Glu Leu Pro Ile Ala Leu Ala Pro Thr Glu140 145 150 155 tgg tac gat atc gag ggt gtt tta tca ggc tca acc acg atcctc cct 533 Trp Tyr Asp Ile Glu Gly Val Leu Ser Gly Ser Thr Thr Ile LeuPro 160 165 170 gat ggt cga atc ttt gct ctc tat acc gga aac aca aac gatctc gag 581 Asp Gly Arg Ile Phe Ala Leu Tyr Thr Gly Asn Thr Asn Asp LeuGlu 175 180 185 caa ctt caa tgc aaa gcc gtg cca gtt aat gca tcc gac ccactt ctt 629 Gln Leu Gln Cys Lys Ala Val Pro Val Asn Ala Ser Asp Pro LeuLeu 190 195 200 gtt gaa tgg gtc agg tac gat gct aac ccg atc ctg tat gctcca tca 677 Val Glu Trp Val Arg Tyr Asp Ala Asn Pro Ile Leu Tyr Ala ProSer 205 210 215 ggg atc ggg tta aca gat tac cgg gac ccg tca aca gtt tggacg ggt 725 Gly Ile Gly Leu Thr Asp Tyr Arg Asp Pro Ser Thr Val Trp ThrGly 220 225 230 235 ccc gat gga aaa cat cgg atg atc ata ggg act aaa cgaaat act aca 773 Pro Asp Gly Lys His Arg Met Ile Ile Gly Thr Lys Arg AsnThr Thr 240 245 250 gga ctc gta ctt gta tac cat acc acc gat ttc aca aactac gta atg 821 Gly Leu Val Leu Val Tyr His Thr Thr Asp Phe Thr Asn TyrVal Met 255 260 265 ttg gac gag ccg ttg cac tcg gtc ccc aac act gat atgtgg gaa tgt 869 Leu Asp Glu Pro Leu His Ser Val Pro Asn Thr Asp Met TrpGlu Cys 270 275 280 gtc gac ctt tac cct gtg tca acg acc aac gat agt gcactt gat gtt 917 Val Asp Leu Tyr Pro Val Ser Thr Thr Asn Asp Ser Ala LeuAsp Val 285 290 295 gcg gcc tat ggt ccg ggt atc aag cat gtg ctt aaa gaaagt tgg gag 965 Ala Ala Tyr Gly Pro Gly Ile Lys His Val Leu Lys Glu SerTrp Glu 300 305 310 315 gga cac gcg atg gac ttt tac tcg atc ggg aca tacgat gca ttt aac 1013 Gly His Ala Met Asp Phe Tyr Ser Ile Gly Thr Tyr AspAla Phe Asn 320 325 330 gat aag tgg aca ccc gat aat ccc gaa cta gac gtcggt atc ggg ttg 1061 Asp Lys Trp Thr Pro Asp Asn Pro Glu Leu Asp Val GlyIle Gly Leu 335 340 345 cgg tgc gat tac gga agg ttc ttt gcg tcg aag agcctc tac gac ccg 1109 Arg Cys Asp Tyr Gly Arg Phe Phe Ala Ser Lys Ser LeuTyr Asp Pro 350 355 360 ttg aag aaa cga aga gtc act tgg ggt tat gtt gcggaa tcc gac agt 1157 Leu Lys Lys Arg Arg Val Thr Trp Gly Tyr Val Ala GluSer Asp Ser 365 370 375 tac gac caa gac gtc tct aga gga tgg gct act atttat aat gtt gca 1205 Tyr Asp Gln Asp Val Ser Arg Gly Trp Ala Thr Ile TyrAsn Val Ala 380 385 390 395 agg acc att gta ctc gat cgg aag act gga acccat cta ctt caa tgg 1253 Arg Thr Ile Val Leu Asp Arg Lys Thr Gly Thr HisLeu Leu Gln Trp 400 405 410 ccg gtg gag gaa atc gag agc ttg aga tcc aacggt cat gaa ttc aaa 1301 Pro Val Glu Glu Ile Glu Ser Leu Arg Ser Asn GlyHis Glu Phe Lys 415 420 425 aat ata aca ctt gag ccg ggc tcg atc att cccctc gac gta ggc tca 1349 Asn Ile Thr Leu Glu Pro Gly Ser Ile Ile Pro LeuAsp Val Gly Ser 430 435 440 gct acg cag ttg gac atc gtt gca aca ttt gaggtg gat caa gag gcg 1397 Ala Thr Gln Leu Asp Ile Val Ala Thr Phe Glu ValAsp Gln Glu Ala 445 450 455 tta aaa gca aca agt gac acg aac gac gaa tacggt tgc acc aca agt 1445 Leu Lys Ala Thr Ser Asp Thr Asn Asp Glu Tyr GlyCys Thr Thr Ser 460 465 470 475 tcg ggt gca gcc aaa ggg gaa gtt ttg gaccat tcg ggg att gca gtt 1493 Ser Gly Ala Ala Lys Gly Glu Val Leu Asp HisSer Gly Ile Ala Val 480 485 490 ctt gcc cac gga acc ctt tcg gag tta actccg gtg tat ttc tac att 1541 Leu Ala His Gly Thr Leu Ser Glu Leu Thr ProVal Tyr Phe Tyr Ile 495 500 505 gct aaa aac acc aag gga ggt gtg gat acacat ttt tgt acg gat aaa 1589 Ala Lys Asn Thr Lys Gly Gly Val Asp Thr HisPhe Cys Thr Asp Lys 510 515 520 cta agg tca tca tat gat tat gat ggt gagaag gtg gtg tat ggc agc 1637 Leu Arg Ser Ser Tyr Asp Tyr Asp Gly Glu LysVal Val Tyr Gly Ser 525 530 535 acc gtc cca gtg ctc gac ggc gaa gaa ttcaca atg agg ata ttg gtg 1685 Thr Val Pro Val Leu Asp Gly Glu Glu Phe ThrMet Arg Ile Leu Val 540 545 550 555 gat cat tcg gtg gtg gag ggg ttt gcacaa ggg gga agg aca gta ata 1733 Asp His Ser Val Val Glu Gly Phe Ala GlnGly Gly Arg Thr Val Ile 560 565 570 acg tca aga gtg tat ccc acg aaa gcaata tac gaa gca gcc aag ctt 1781 Thr Ser Arg Val Tyr Pro Thr Lys Ala IleTyr Glu Ala Ala Lys Leu 575 580 585 ttc gtc ttc aac aat gcc act acg accagt gtg aag gcg act ctc aag 1829 Phe Val Phe Asn Asn Ala Thr Thr Thr SerVal Lys Ala Thr Leu Lys 590 595 600 gtc tgg caa atg tct caa gcc ttt gtcaag gct tat ccg ttt 1871 Val Trp Gln Met Ser Gln Ala Phe Val Lys Ala TyrPro Phe 605 610 615 tagtttttta tgcatctttt taagacattg ttgtttcatatgattcaagt tttatctgtg 1931 tgttatgtta agacacgcag cttaaaatag ccacatgtgagatcatttgc gtatggccgt 1991 caactatttt ttaatatgca acttcagtaa tgctatttacagtatgtttt aaggaaaaaa 2051 aaaaaaaaaa aaaaaaaaaa aa 2073 2 617 PRTCynara scolymus 2 Met Arg Thr Thr Glu Pro Gln Thr Asp Leu Glu His AlaPro Asn His 1 5 10 15 Thr Pro Leu Leu Asp His Pro Glu Pro Pro Pro AlaAla Val Arg Asn 20 25 30 Arg Leu Leu Ile Arg Val Ser Ser Ser Ile Thr LeuVal Ser Leu Phe 35 40 45 Phe Val Ser Ala Phe Leu Leu Ile Leu Leu Tyr GlnHis Asp Ser Thr 50 55 60 Tyr Thr Asp Asp Asn Ser Ala Pro Ser Glu Ser SerSer Gln Gln Pro 65 70 75 80 Ser Ala Ala Asp Arg Leu Arg Trp Glu Arg ThrAla Phe His Phe Gln 85 90 95 Pro Ala Lys Asn Phe Ile Tyr Asp Pro Asn GlyPro Leu Phe His Met 100 105 110 Gly Trp Tyr His Leu Phe Tyr Gln Tyr AsnPro Tyr Ala Pro Phe Trp 115 120 125 Gly Asn Met Thr Trp Gly His Ala ValSer Lys Asp Met Ile Asn Trp 130 135 140 Phe Glu Leu Pro Ile Ala Leu AlaPro Thr Glu Trp Tyr Asp Ile Glu 145 150 155 160 Gly Val Leu Ser Gly SerThr Thr Ile Leu Pro Asp Gly Arg Ile Phe 165 170 175 Ala Leu Tyr Thr GlyAsn Thr Asn Asp Leu Glu Gln Leu Gln Cys Lys 180 185 190 Ala Val Pro ValAsn Ala Ser Asp Pro Leu Leu Val Glu Trp Val Arg 195 200 205 Tyr Asp AlaAsn Pro Ile Leu Tyr Ala Pro Ser Gly Ile Gly Leu Thr 210 215 220 Asp TyrArg Asp Pro Ser Thr Val Trp Thr Gly Pro Asp Gly Lys His 225 230 235 240Arg Met Ile Ile Gly Thr Lys Arg Asn Thr Thr Gly Leu Val Leu Val 245 250255 Tyr His Thr Thr Asp Phe Thr Asn Tyr Val Met Leu Asp Glu Pro Leu 260265 270 His Ser Val Pro Asn Thr Asp Met Trp Glu Cys Val Asp Leu Tyr Pro275 280 285 Val Ser Thr Thr Asn Asp Ser Ala Leu Asp Val Ala Ala Tyr GlyPro 290 295 300 Gly Ile Lys His Val Leu Lys Glu Ser Trp Glu Gly His AlaMet Asp 305 310 315 320 Phe Tyr Ser Ile Gly Thr Tyr Asp Ala Phe Asn AspLys Trp Thr Pro 325 330 335 Asp Asn Pro Glu Leu Asp Val Gly Ile Gly LeuArg Cys Asp Tyr Gly 340 345 350 Arg Phe Phe Ala Ser Lys Ser Leu Tyr AspPro Leu Lys Lys Arg Arg 355 360 365 Val Thr Trp Gly Tyr Val Ala Glu SerAsp Ser Tyr Asp Gln Asp Val 370 375 380 Ser Arg Gly Trp Ala Thr Ile TyrAsn Val Ala Arg Thr Ile Val Leu 385 390 395 400 Asp Arg Lys Thr Gly ThrHis Leu Leu Gln Trp Pro Val Glu Glu Ile 405 410 415 Glu Ser Leu Arg SerAsn Gly His Glu Phe Lys Asn Ile Thr Leu Glu 420 425 430 Pro Gly Ser IleIle Pro Leu Asp Val Gly Ser Ala Thr Gln Leu Asp 435 440 445 Ile Val AlaThr Phe Glu Val Asp Gln Glu Ala Leu Lys Ala Thr Ser 450 455 460 Asp ThrAsn Asp Glu Tyr Gly Cys Thr Thr Ser Ser Gly Ala Ala Lys 465 470 475 480Gly Glu Val Leu Asp His Ser Gly Ile Ala Val Leu Ala His Gly Thr 485 490495 Leu Ser Glu Leu Thr Pro Val Tyr Phe Tyr Ile Ala Lys Asn Thr Lys 500505 510 Gly Gly Val Asp Thr His Phe Cys Thr Asp Lys Leu Arg Ser Ser Tyr515 520 525 Asp Tyr Asp Gly Glu Lys Val Val Tyr Gly Ser Thr Val Pro ValLeu 530 535 540 Asp Gly Glu Glu Phe Thr Met Arg Ile Leu Val Asp His SerVal Val 545 550 555 560 Glu Gly Phe Ala Gln Gly Gly Arg Thr Val Ile ThrSer Arg Val Tyr 565 570 575 Pro Thr Lys Ala Ile Tyr Glu Ala Ala Lys LeuPhe Val Phe Asn Asn 580 585 590 Ala Thr Thr Thr Ser Val Lys Ala Thr LeuLys Val Trp Gln Met Ser 595 600 605 Gln Ala Phe Val Lys Ala Tyr Pro Phe610 615 3 2073 DNA Cynara scolymus CDS (21)..(1871) 3 ttacctcatttccatcaacc atg aga acg act gaa ccc caa act gac ctt gag 53 Met Arg ThrThr Glu Pro Gln Thr Asp Leu Glu 1 5 10 cat gca ccc aac cac act cca ctactg gac cac ccc gaa cca cca ccg 101 His Ala Pro Asn His Thr Pro Leu LeuAsp His Pro Glu Pro Pro Pro 15 20 25 gcc gcc gtg aga aac cgg ttg ttg attagg gtt tcg tcc agt atc aca 149 Ala Ala Val Arg Asn Arg Leu Leu Ile ArgVal Ser Ser Ser Ile Thr 30 35 40 ttg gtc tct ctg ttt ttt gtt tca gca ttccta ctc att ctc ctg tac 197 Leu Val Ser Leu Phe Phe Val Ser Ala Phe LeuLeu Ile Leu Leu Tyr 45 50 55 caa cac gat tcc act tac acc gat gat aat tcagca ccg tcg gaa agt 245 Gln His Asp Ser Thr Tyr Thr Asp Asp Asn Ser AlaPro Ser Glu Ser 60 65 70 75 tct tcc cag cag ccc tcc gct gcc gat cgc ctgaga tgg gag aga aca 293 Ser Ser Gln Gln Pro Ser Ala Ala Asp Arg Leu ArgTrp Glu Arg Thr 80 85 90 gct ttt cat ttc cag ccc gcc aaa aat ttc att tatgat ccc aac ggt 341 Ala Phe His Phe Gln Pro Ala Lys Asn Phe Ile Tyr AspPro Asn Gly 95 100 105 cca ttg ttc cat atg ggt tgg tac cat ctt ttc taccaa tac aac ccg 389 Pro Leu Phe His Met Gly Trp Tyr His Leu Phe Tyr GlnTyr Asn Pro 110 115 120 tac gct ccc ttt tgg gga aac atg act tgg gga catgcc gtc agt aag 437 Tyr Ala Pro Phe Trp Gly Asn Met Thr Trp Gly His AlaVal Ser Lys 125 130 135 gat atg ata aat tgg ttt gaa tta ccg ata gcc ttagcg cca act gag 485 Asp Met Ile Asn Trp Phe Glu Leu Pro Ile Ala Leu AlaPro Thr Glu 140 145 150 155 tgg tac gac ata gaa ggt gtt ctg agt ggc agtact acc att tta cct 533 Trp Tyr Asp Ile Glu Gly Val Leu Ser Gly Ser ThrThr Ile Leu Pro 160 165 170 gac gga aga att ttc gct ctc tac acc gga aataca aac gac ctc gag 581 Asp Gly Arg Ile Phe Ala Leu Tyr Thr Gly Asn ThrAsn Asp Leu Glu 175 180 185 cag ctc cag tgt aag gcc gtg cca gtt aat gctagt gat cca tta ttg 629 Gln Leu Gln Cys Lys Ala Val Pro Val Asn Ala SerAsp Pro Leu Leu 190 195 200 gta gaa tgg gtt cgc tac gat gcc aat ccg atatta tat gcc cct agt 677 Val Glu Trp Val Arg Tyr Asp Ala Asn Pro Ile LeuTyr Ala Pro Ser 205 210 215 ggc atc ggc ctc aca gat tac aga gat cct agtact gtg tgg acg ggc 725 Gly Ile Gly Leu Thr Asp Tyr Arg Asp Pro Ser ThrVal Trp Thr Gly 220 225 230 235 cct gac ggt aaa cac cgt atg ata atc gggacg aag agg aat acg act 773 Pro Asp Gly Lys His Arg Met Ile Ile Gly ThrLys Arg Asn Thr Thr 240 245 250 gga ctc gtc tta gta tat cac act acc gacttt aca aat tat gta atg 821 Gly Leu Val Leu Val Tyr His Thr Thr Asp PheThr Asn Tyr Val Met 255 260 265 ttg gac gag ccg ttg cac tcg gtc ccc aacact gat atg tgg gaa tgt 869 Leu Asp Glu Pro Leu His Ser Val Pro Asn ThrAsp Met Trp Glu Cys 270 275 280 gtc gac ctt tac cct gtg tca acg acc aacgat agt gca ctt gat gtt 917 Val Asp Leu Tyr Pro Val Ser Thr Thr Asn AspSer Ala Leu Asp Val 285 290 295 gcg gcc tat ggt ccg ggt atc aag cat gtgctt aaa gaa agt tgg gag 965 Ala Ala Tyr Gly Pro Gly Ile Lys His Val LeuLys Glu Ser Trp Glu 300 305 310 315 gga cac gcg atg gac ttt tac tcg atcggg aca tac gat gca ttt aac 1013 Gly His Ala Met Asp Phe Tyr Ser Ile GlyThr Tyr Asp Ala Phe Asn 320 325 330 gat aag tgg aca ccc gat aat ccc gaacta gac gtc ggt atc ggg ttg 1061 Asp Lys Trp Thr Pro Asp Asn Pro Glu LeuAsp Val Gly Ile Gly Leu 335 340 345 cgg tgc gat tac gga agg ttc ttt gcgtcg aag agc ctc tac gac ccg 1109 Arg Cys Asp Tyr Gly Arg Phe Phe Ala SerLys Ser Leu Tyr Asp Pro 350 355 360 ttg aag aaa cga aga gtc act tgg ggttat gtt gcg gaa tcc gac agt 1157 Leu Lys Lys Arg Arg Val Thr Trp Gly TyrVal Ala Glu Ser Asp Ser 365 370 375 tac gac caa gac gtc tct aga gga tgggct act att tat aat gtt gca 1205 Tyr Asp Gln Asp Val Ser Arg Gly Trp AlaThr Ile Tyr Asn Val Ala 380 385 390 395 agg acc att gta ctc gat cgg aagact gga acc cat cta ctt caa tgg 1253 Arg Thr Ile Val Leu Asp Arg Lys ThrGly Thr His Leu Leu Gln Trp 400 405 410 ccg gtg gag gaa atc gag agc ttgaga tcc aac ggt cat gaa ttc aaa 1301 Pro Val Glu Glu Ile Glu Ser Leu ArgSer Asn Gly His Glu Phe Lys 415 420 425 aat ata aca ctt gag ccg ggc tcgatc att ccc ctc gac gta ggc tca 1349 Asn Ile Thr Leu Glu Pro Gly Ser IleIle Pro Leu Asp Val Gly Ser 430 435 440 gct acg cag ttg gac atc gtt gcaaca ttt gag gtg gat caa gag gcg 1397 Ala Thr Gln Leu Asp Ile Val Ala ThrPhe Glu Val Asp Gln Glu Ala 445 450 455 tta aaa gca aca agt gac acg aacgac gaa tac ggt tgc acc aca agt 1445 Leu Lys Ala Thr Ser Asp Thr Asn AspGlu Tyr Gly Cys Thr Thr Ser 460 465 470 475 tcg ggt gca gcc aaa ggg gaagtt ttg gac cat tcg ggg att gca gtt 1493 Ser Gly Ala Ala Lys Gly Glu ValLeu Asp His Ser Gly Ile Ala Val 480 485 490 ctt gcc cac gga acc ctt tcggag tta act ccg gtg tat ttc tac att 1541 Leu Ala His Gly Thr Leu Ser GluLeu Thr Pro Val Tyr Phe Tyr Ile 495 500 505 gct aaa aac acc aag gga ggtgtg gat aca cat ttt tgt acg gat aaa 1589 Ala Lys Asn Thr Lys Gly Gly ValAsp Thr His Phe Cys Thr Asp Lys 510 515 520 cta agg tca tca tat gat tatgat ggt gag aag gtg gtg tat ggc agc 1637 Leu Arg Ser Ser Tyr Asp Tyr AspGly Glu Lys Val Val Tyr Gly Ser 525 530 535 acc gtc cca gtg ctc gac ggcgaa gaa ttc aca atg agg ata ttg gtg 1685 Thr Val Pro Val Leu Asp Gly GluGlu Phe Thr Met Arg Ile Leu Val 540 545 550 555 gat cat tcg gtg gtg gagggg ttt gca caa ggg gga agg aca gta ata 1733 Asp His Ser Val Val Glu GlyPhe Ala Gln Gly Gly Arg Thr Val Ile 560 565 570 acg tca aga gtg tat cccacg aaa gca ata tac gaa gca gcc aag ctt 1781 Thr Ser Arg Val Tyr Pro ThrLys Ala Ile Tyr Glu Ala Ala Lys Leu 575 580 585 ttc gtc ttc aac aat gccact acg acc agt gtg aag gcg act ctc aag 1829 Phe Val Phe Asn Asn Ala ThrThr Thr Ser Val Lys Ala Thr Leu Lys 590 595 600 gtc tgg caa atg tct caagcc ttt gtc aag gct tat ccg ttt 1871 Val Trp Gln Met Ser Gln Ala Phe ValLys Ala Tyr Pro Phe 605 610 615 tagtttttta tgcatctttt taagacattgttgtttcata tgattcaagt tttatctgtg 1931 tgttatgtta agacacgcag cttaaaatagccacatgtga gatcatttgc gtatggccgt 1991 caactatttt ttaatatgca acttcagtaatgctatttac agtatgtttt aaggaaaaaa 2051 aaaaaaaaaa aaaaaaaaaa aa 2073 4617 PRT Cynara scolymus 4 Met Arg Thr Thr Glu Pro Gln Thr Asp Leu GluHis Ala Pro Asn His 1 5 10 15 Thr Pro Leu Leu Asp His Pro Glu Pro ProPro Ala Ala Val Arg Asn 20 25 30 Arg Leu Leu Ile Arg Val Ser Ser Ser IleThr Leu Val Ser Leu Phe 35 40 45 Phe Val Ser Ala Phe Leu Leu Ile Leu LeuTyr Gln His Asp Ser Thr 50 55 60 Tyr Thr Asp Asp Asn Ser Ala Pro Ser GluSer Ser Ser Gln Gln Pro 65 70 75 80 Ser Ala Ala Asp Arg Leu Arg Trp GluArg Thr Ala Phe His Phe Gln 85 90 95 Pro Ala Lys Asn Phe Ile Tyr Asp ProAsn Gly Pro Leu Phe His Met 100 105 110 Gly Trp Tyr His Leu Phe Tyr GlnTyr Asn Pro Tyr Ala Pro Phe Trp 115 120 125 Gly Asn Met Thr Trp Gly HisAla Val Ser Lys Asp Met Ile Asn Trp 130 135 140 Phe Glu Leu Pro Ile AlaLeu Ala Pro Thr Glu Trp Tyr Asp Ile Glu 145 150 155 160 Gly Val Leu SerGly Ser Thr Thr Ile Leu Pro Asp Gly Arg Ile Phe 165 170 175 Ala Leu TyrThr Gly Asn Thr Asn Asp Leu Glu Gln Leu Gln Cys Lys 180 185 190 Ala ValPro Val Asn Ala Ser Asp Pro Leu Leu Val Glu Trp Val Arg 195 200 205 TyrAsp Ala Asn Pro Ile Leu Tyr Ala Pro Ser Gly Ile Gly Leu Thr 210 215 220Asp Tyr Arg Asp Pro Ser Thr Val Trp Thr Gly Pro Asp Gly Lys His 225 230235 240 Arg Met Ile Ile Gly Thr Lys Arg Asn Thr Thr Gly Leu Val Leu Val245 250 255 Tyr His Thr Thr Asp Phe Thr Asn Tyr Val Met Leu Asp Glu ProLeu 260 265 270 His Ser Val Pro Asn Thr Asp Met Trp Glu Cys Val Asp LeuTyr Pro 275 280 285 Val Ser Thr Thr Asn Asp Ser Ala Leu Asp Val Ala AlaTyr Gly Pro 290 295 300 Gly Ile Lys His Val Leu Lys Glu Ser Trp Glu GlyHis Ala Met Asp 305 310 315 320 Phe Tyr Ser Ile Gly Thr Tyr Asp Ala PheAsn Asp Lys Trp Thr Pro 325 330 335 Asp Asn Pro Glu Leu Asp Val Gly IleGly Leu Arg Cys Asp Tyr Gly 340 345 350 Arg Phe Phe Ala Ser Lys Ser LeuTyr Asp Pro Leu Lys Lys Arg Arg 355 360 365 Val Thr Trp Gly Tyr Val AlaGlu Ser Asp Ser Tyr Asp Gln Asp Val 370 375 380 Ser Arg Gly Trp Ala ThrIle Tyr Asn Val Ala Arg Thr Ile Val Leu 385 390 395 400 Asp Arg Lys ThrGly Thr His Leu Leu Gln Trp Pro Val Glu Glu Ile 405 410 415 Glu Ser LeuArg Ser Asn Gly His Glu Phe Lys Asn Ile Thr Leu Glu 420 425 430 Pro GlySer Ile Ile Pro Leu Asp Val Gly Ser Ala Thr Gln Leu Asp 435 440 445 IleVal Ala Thr Phe Glu Val Asp Gln Glu Ala Leu Lys Ala Thr Ser 450 455 460Asp Thr Asn Asp Glu Tyr Gly Cys Thr Thr Ser Ser Gly Ala Ala Lys 465 470475 480 Gly Glu Val Leu Asp His Ser Gly Ile Ala Val Leu Ala His Gly Thr485 490 495 Leu Ser Glu Leu Thr Pro Val Tyr Phe Tyr Ile Ala Lys Asn ThrLys 500 505 510 Gly Gly Val Asp Thr His Phe Cys Thr Asp Lys Leu Arg SerSer Tyr 515 520 525 Asp Tyr Asp Gly Glu Lys Val Val Tyr Gly Ser Thr ValPro Val Leu 530 535 540 Asp Gly Glu Glu Phe Thr Met Arg Ile Leu Val AspHis Ser Val Val 545 550 555 560 Glu Gly Phe Ala Gln Gly Gly Arg Thr ValIle Thr Ser Arg Val Tyr 565 570 575 Pro Thr Lys Ala Ile Tyr Glu Ala AlaLys Leu Phe Val Phe Asn Asn 580 585 590 Ala Thr Thr Thr Ser Val Lys AlaThr Leu Lys Val Trp Gln Met Ser 595 600 605 Gln Ala Phe Val Lys Ala TyrPro Phe 610 615

We claim:
 1. An isolated nucleic acid molecule comprising a acidsequence encoding a fructosyl transferase (FFT) protein, wherein thenucleic acid sequence is selected from the group consisting of: (a) anucleic acid sequence encoding a protein comprising the amino acidsequence of SEQ ID NO: 2 or SEQ ID NO: 4; (b) a nucleic acid sequencecomprising the coding region of SEQ ID NO: 1 or SEQ ID NO: 3; (c) anucleic acid sequence that hybridizes to a complementary strand of thenucleic acid sequence named under (a) or (b) under conditions ofhybridization in 50% formamide, 5×SSC, 5×Denhardt's solution, 40 mMsodium phosphate pH 6.8; 0.5% (w/v) BSA, 1% (w/v) SDS and 0.1 mg/mlherring sperm DNA at 42° C. and washing in 0.5×SSC/0.5% SDS at 60° C.;and (d) a nucleic acid sequence comprising a fragment of the nucleotidesequence of (a), (b) or (c), said fragment being of a length sufficientto encode a FFT protein.
 2. The nucleic acid molecule according to claim1, wherein the molecule is a DNA molecule.
 3. The nucleic acid moleculeaccording to claim 2, wherein the molecule is a cDNA molecule.
 4. Thenucleic acid molecule according to claim 1, wherein the molecule is anRNA molecule.
 5. The nucleic acid molecule according to any one ofclaims 1 to 4 that is derived from artichoke.
 6. An isolated nucleicacid molecule comprising a nucleic acid sequence selected from the groupconsisting of the coding region of SEQ ID NO: 1, the coding region ofSEQ ID NO: 3, a nucleic acid sequence encoding a protein comprising theamino acid sequence of SEQ ID NO: 2, and a nucleic acid sequenceencoding a protein comprising the amino acid sequence of SEQ ID NO: 4.7. An isolated nucleic acid molecule comprising a nucleic acid sequenceencoding a fructosyl transferase (FFT) protein, wherein the nucleic acidsequence is more than 80% identical to a nucleic acid sequence selectedfrom the group consisting of: (a) a nucleic acid sequence encoding aprotein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:4; (b) the coding region of SEQ ID NO: 1 or SEQ ID NO: 3; and (c) anucleic acid sequence comprising a fragment of the nucleotide sequenceof (a) or (b), wherein the fragment encodes a fructosyl transferase(FFT) protein.
 8. An isolated nucleic acid molecule comprising a nucleicacid sequence encoding a fructosyl transferase (FFT) protein, whereinthe nucleic acid sequence is more than 90% identical to a nucleic acidsequence selected from the group consisting of: (a) a nucleic acidsequence encoding a protein comprising the amino acid sequence of SEQ IDNO: 2 or SEQ ID NO: 4; (b) the coding region of SEQ ID NO: 1 or SEQ IDNO: 3; and (c) a nucleic acid sequence comprising a fragment of thenucleotide sequence of (a) or (b), wherein the fragment encodes afructosyl transferase (FFT) protein.
 9. A vector comprising the nucleicacid molecule according to any one of claims 1-4 or 6-8.
 10. The vectoraccording to claim 9, wherein the nucleic acid molecule is operativelylinked to a regulatory element ensuring the transcription and synthesisof a translatable RNA in a prokaryotic cell a eukaryotic cell, or aprocaryotic and eukaryotic cell.
 11. The vector according to claim 10,wherein the regulatory element is derived from the patatin B33 promoteror the CaMV 35S promoter.
 12. A host cell transformed with the nucleicacid molecule according to any one of claims 1-4 or 6-8, or a host cellthat comprises a vector comprising the nucleic acid molecule, or aderivative of said host cell, wherein said derivative comprises saidnucleic acid molecule.
 13. The host cell according to claim 12, whereinthe cell additionally comprises a nucleic acid molecule comprising anucleic acid sequence encoding a sucrose-dependent sucrose fructosyltransferase (SST).
 14. A method for the production of an FFT, comprisingthe steps of cultivating the host cell according to claim 12 underconditions allowing for the synthesis of the FFT.
 15. An isolatednucleic acid molecule that specifically hybridizes to the nucleic acidmolecule according to claim 6 or to a complementary strand thereof underconditions of hybridization in 50% formamide, 5×SSC. 5×Denhardt'ssolution 40 mM sodium phosphate pH 6.8; 0.5% (w/v) BSA, 1% (w/v) SDS and0.1 mg/ml herring sperm DNA at 42 degrees C and washing in 0.5×SSC/0.5%SDS at 60 degrees C.
 16. A method for the production of a host cell, atransgenic plant cell, a transgenic plant tissue or a transgenic plant,comprising the step of introducing the nucleic acid molecule accordingto any one of claims 1-4 or 6-8, or a vector comprising said nucleicacid molecule, into the host cell, the plant cell, the plant tissue orthe plant.
 17. A transgenic plant cell or plant tissue that comprisesthe nucleic acid molecule according to any one of claims 1-4 or 6-8, orthat comprises a vector comprising said nucleic acid molecule, or aderivative of said cell or tissue, wherein said derivative comprisessaid nucleic acid molecule.
 18. The transgenic plant cell, plant tissueor plant according to claim 17, additionally comprising a nucleic acidmolecule comprising a nucleic acid sequence that encodes asucrose-dependent sucrose fructosyl transferase.
 19. A plant comprisingthe plant cell or plant tissue according to claim 17, or comprising saidplant cell or said plant tissue that additionally comprises a geneencoding an SST.
 20. The plant according to claim 19, wherein the plantis a sucrose-containing plant.
 21. The plant according to claim 20,wherein the sucrose-containing plant is selected from the groupconsisting of a rice, maize, sugar beet, sugar cane, potato, tomato,carrot, leek, chicory, sweet potato, wheat, barley, rape and soybeanplant.
 22. A propagation material of a plant comprising the plant cellaccording to claim 15 or comprising said plant cell that additionallycomprises a gene encoding an SST.
 23. A harvest product of a plantcomprising a plant cell according to claim 17 or comprising said plantcell that additionally comprises a gene encoding an SST.
 24. A methodfor the production of high molecular inulin comprising (a) cultivatingthe host cell according to claim 12; or a transgenic plant cell, planttissue or plant comprising said host cell; or said host cell, transgenicplant cell, plant tissue or plant that additionally comprise a geneencoding SST, under conditions which allow for the production of an FFTand the conversion of 1-kestose or an equivalent substrate into highmolecular inulin, wherein 1-kestose or the equivalent substrate isoptionally added to the culture, and (b) recovering the thus producedinulin from the cultivated cell, tissue or plant or from a medium inwhich the cell or tissue may have been cultivated.
 25. The isolatednucleic acid molecule according to claim 6, wherein the nucleic acidsequence is SEQ ID NO: 1 or SEQ ID NO:
 3. 26. The vector according toclaim 9, wherein the nucleic acid molecule is derived from artichoke.27. The host cell according to claim 12, wherein the nucleic acidmolecule is derived from artichoke.
 28. The method according to claim16, wherein the nucleic acid molecule is derived from artichoke.
 29. Thetransgenic plant cell or plant tissue according to claim 17, wherein thenucleic acid molecule is derived from artichoke.